:: Asian Journal of Andrology ::

Effect of follicle-stimulating hormone on sperm quality and pregnancy rate

B. Baccetti¹, P. Piomboni¹, E. Bruni¹, S. Capitani¹, L. Gambera¹, E. Moretti¹, K. Sterzik¹, E. Strehler²

¹Department of Paediatrics, Obstetrics and Reproductive Medicine, Section of Biology, University of Siena, Regional Referral Center for Male Infertility, Azienda Ospedaliera Senese, Siena, Italy
²Institute for Reproductive Medicine, Ulm, Germany

Asian J Androl 2004 Jun; 6: 133-137

Keywords: male infertility; spermatozoa; electron microscopy; FSH therapy; clinical pregnancy

Abstract

Aim: To evaluate the possible links between ultrastructural sperm quality and the clinical pregnancy rate in infertile males treated with FSH before intracytoplasmic sperm injection (ICSI). Methods: Forty-four infertile males with idiopathic oligo-asthenozoospermia were randomly allocated to the treated (n=24) and non-treated (control, n=20) groups. Semen analysis was carried out by light and transmission electron microscopy (TEM) before and 12 weeks after FSH therapy. ICSI was performed in all couples. Results: TEM revealed a significant improvement in sperm quality after FSH treatment, particularly in men with their partners achieving clinical pregnancy. The pregnancy rate was 33 % in the treated group and 20 % in the control. Conclusion: Results highlight a positive role of FSH therapy in infertile males before ICSI, which was correlated with an increased pregnancy rate in treated couples. We believe that improved sperm ultrastructure after FSH therapy could positively influence the quality and early stage of embryo development, thereby increasing the probability of embryo implantation.

1 Introduction

The efficacy of FSH in the treatment of male infertility is a controversial question with various reports drawing different conclusion. Improved male germinal cell quality and fertilizing ability after FSH treatment has been highlighted by Acosta et al [1, 2] and Foresta et al [3]; statistical evaluation based on sperm ultrastructure has been done by Strehler et al [4], Baccetti et al [5] and Ben-Rafael et al [6]. More recently, Foresta et al [7] demonstrated that r-hFSH therapy increased the spermatogonial population and sperm production in patients with idiopathic oligozoospermia. Systemic administration of purified FSH to infertile males has also been tested; Kamischke et al [8] failed to find any improvement in sperm quality or pregnancy rate in the treated group; whereas Ashkenazi et al [9, 10] reported improvement in pregnancy rate after intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI).

The aim of the present study was to evaluate the possible links between ultrastructural sperm quality and the clinical pregnancy rate in infertile males treated with FSH before intracytoplasmic sperm injection (ICSI).

2 Materials and methods

2.1 Patients

The subjects of this study were 44 couples attending the Institute of Reproductive Medicine in Ulm, Germany, for infertility. The results of combined gynaecological and andrological examinations indicated idiopathic male factor infertility in all couples. The female partners (mean age 32 years) underwent a complete infertility workup. No health or fertility problems were found. None of the women had untreatable hormonal irregularities.

The inclusion criteria for male partners were: a) normal or low baseline endocrine parameters, including plasma concentrations of FSH, LH, prolactin, 17-estradiol and testosterone; b) no causes of infertility detected by physical examinations, such as reduced testicular size, varicocele, cryptorchidism, etc; c) absence of urinogenital tract infection or inflammation; d) unremarkable medical history; e) oligo- and/or astheno-zoospermia according to WHO criteria [11]; f) failure in at least two in vitro fertilization (IVF) or IUI treatment cycles. Patients with plasma concentrations of FSH above 12 mUI/mL were excluded from the study. Written informed consent was obtained from all patients.

2.2 Treatment protocol

The 44 males (age 28 to 45 years) recruited were allocated either to the treated (n=24) or non-treated (control, n=20) group by third party true randomisation. The different size between the two groups was established at the beginning of the treatment. The treated group received FSH (Fertinorm HP, Serono, Unterschleissheim, Germany) at 150 IU/day s.c. for 12 weeks.

2.3 Semen analysis

All patients underwent semen analysis by optical microscopy according to WHO guidelines [11] before and after FSH treatment. Semen samples were obtained after 4 days of sexual abstinence and liquefied at 37 . Sperm count and motility were assessed using a Makler counting chamber.

2.4 Transmission electron microscopy

Aliquots of each semen sample were examined by transmission electron microscopy (TEM). Spermatozoa were fixed in Karnovsky fixative, rinsed overnight in 0.1 mol/L cacodylate buffer (pH 7.2), postfixed in 1 % buffered OsO₄, dehydrated and embedded in Epon Araldite. Ultrathin sections were cut with an LKB ultramicrotome (Wien, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a CM 10 transmission electron microscope (Philips, Eindhoven, The Netherlands) at magnifications of 15 000 to 75 000. Two highly trained researchers, who did not know who had been treated, did the TEM analysis.

TEM data was evaluated using the mathematical formula of Baccetti et al [12] to check for improvement in sperm ultrastructural characteristics. The mean total numbers and mean percentages of sperm devoid of defects ("healthy") between the two groups were statistically compared. Moreover, the mean percentages of the three more usual sperm pathology (namely immaturity, necrosis, apoptosis) diagnosed in the ejaculate were compared before and after the therapy.

2.5 ICSI procedure and embryo transfer

Hormonal stimulation with hMG and hCG, sono-graphic monitoring of follicular growth, oocyte aspiration and oocyte culture were performed according to previously described protocols [13, 14]. To perform ICSI, an inverted microscope (Leitz Diavert, Wetzlar, Germany) equipped with Narishige micromanipulators and a 37 heating stage were used. ICSI was performed as described by Van Steirteghem [15, 16]. Up to three embryos were transferred to the uterine cavity two days after oocyte retrieval. Clinical pregnancy was determined by ultrasound evidence of a foetal sac six weeks after embryo transfer.

2.6 Statistical analysis

Data were in meanSD if applicable. The Student's t-test and the signed rank test were used as appropriate. P < 0.05 was considered statistically significant.

3 Results

Spermiograms of all males before treatment revealed oligoasthenozoospermia (Table 1). No significant differences of sperm parameters were observed between the treated and control groups. There was no improvement in sperm count or progressive motility observed in the FSH treated group, nor any change in seminal parameters in the control group. Nevertheless, FSH treatment appeared to significantly improve sperm ultrastructural characteristics (Figure 1a, b), with significantly increased mean total number and mean percentage of healthy sperm after therapy (signed rank test ^bP<0.05 and ^cP<0.01, respectively; Table 2). Ultrastructural sperm characteristics improved in the eight couples achieving pregnancy after FSH therapy with significantly improved mean percentages of healthy sperm after treatment (signed rank test ^bP < 0.05; t-test ^eP < 0.05; Table 3). As far as the three most common sperm pathologies were concerned, immaturity was much less frequent after treatment; apoptosis and necrosis were also less frequent, though not significantly (Table 4). In the control group, there was no improvement in ultrastructural sperm quality of the four couples achieving pregnancy after ICSI. The mean number of oocytes retrieved, the percentage of oocytes fertilized and the mean number of embryos transferred were very similar in the two groups. However, the pregnancy rate was 33 % in the treated group and only 20 % in the controls (P < 0.05, Table 5).

Figure 1a. Nuclei with abnormal morphology, showing marginated chromatin (mch) and altered acrosomes (aA). Note large cytoplasmic residues (RC) sometimes with translucent vacuoles. In some cases, the plasma membrane appears broken (bPM). An apoptotic body (aB) is visible among the sperm (6200).
Figure 1b. Many sperm have nuclei (nN) and acrosomes (nA) with normal morphology (11 000).
Figure 1. Longitudinal and cross sections of ejaculated spermatozoa before (a) and after (b) FSH treatment of males with their partners achieving clinical pregnancy.

Table 1. Semen analysis by light microscopy according to WHO guidelines [11].

	Treated patients		Control patients
	Before FSH	After FSH	Non-treatment	Non-treatment
	treatment (n=24)	treatment (n= 24)	before (n=20)	after (n=20)
Semen volume (mL)	3.30.2	3.00.9	4.41.1	4.20.5
Sperm count (10⁶/mL)	8.81.7	8.91.2	12.42.1	12.73.2
Progressive motility (%)	28.02.5	26.51.9	32.01.7	35.02.2

Table 2. Effect of FSH therapy on sperm quality evaluated by electron microscopy. Signed rank test: ^bP<0.05, Signed rank test: ^cP<0.01,compared with before FSH treatment.

	Treated patients		Control patients
	Before FSH	After FSH	Non-treatment	Non-treatment
	treatment (n=24)	treatment (n=24)	before (n=20)	after (n=20)
Mean number of sperm without defects	26 999	111 517^b	23 812	28 412
Mean percentage of sperm without defects (%)	0.0007	0.0033^c	0.0008	0.05%

Table 3. Effect of FSH therapy on sperm quality evaluated by electron microscopy in males with their partners achieving clinical pregnancy. Signed rank test: ^bP<0.05, t-test: ^eP<0.05, compared with before FSH treatment.

	Before FSH	After FSH
	treatment (n=8)	treatment (n= 8)
Mean number of sperm without defects	30 960	250 979
Percentage of sperm without defects (%)	0.05	0.63^b,e

Table 4. Mean percentages of sperm pathology in males, whose partners achieved clinical pregnancy after ICSI. ^aP>0.05, compared with pre-treatment value.

Sperm pathology	Before FSH treatment (n=8)	After FSH treatment (n= 8)
Immaturity (%)	74.03	61.94^a
Necrosis (%)	49.66	33.15^a
Apoptosis (%)	17.54	7.83^a

Table 5. Clinical outcome of ICSI in treatment and control groups. ^aP>0.05, compared with the controls.

	Treatment group (n=24)	Control group (n=20)
Mean number of oocytes retrieved	7.53.0	6.32.6
Fertilization rate (%)	47	37
Mean number of embryos transferred	2.50.5	2.40.9
Clinical pregnancy rate (%)	33^a	20
Miscarriage	1	1

4 Discussion

FSH therapy of male partners before ICSI gave positive results, as demonstrated by the significant improvement in sperm ultrastructural characteristics and marked increase in pregnancy rate in treated (33 %) versus non-treated (20 %) couples. The clinical pregnancy rate in treated couples was also much higher than the mean pregnancy rate in European countries reported by ESHRE [17]. Unfortunately, since no data on the outcome of pregnancies is available, no conclusion on the relationship between this parameter and the improvement in sperm ultrastructure can be drawn.

The link between the morphological sperm quality and pregnancy rate after ICSI is not a new finding. Strehler et al [4] and Bartoov et al [18] using electron microscopy and Nomarski microscopy, respectively, reported a significant correlation between the sperm quality and embryo number or pregnancy rate after ICSI. The improvement of sperm structure supports previous results obtained by Baccetti et al [5], Ashkenazi et al [9, 10], Ben Rafael et al [6] and Foresta [3, 7]. Haidl et al [19] suggested that the administration of a high-dose FSH is particularly promising for improving the disturbed sperm structure. Moreover, Liu and Baker [20] affirmed that the conventional semen analysis do not provide accurate information about sperm fertilizing ability since many subtle sperm defects can not be detected. In this study, we are able to elucidate the relationship between sperm structure and function by electron microscopy, also find a link with the clinical pregnancy rate.

In conclusion, the ultrastructural characteristics of sperm could be a major factor for embryo survival after ICSI, indicating a possible paternal contribution to embryonic development; FSH therapy before ICSI seems to be a valid treatment for couples with male infertility factor.

Acknowledgments

This study was partly financed by a grant from the Italian Ministry for Universities and Technological Research, 2000.

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Correspondence to: Dr. B. Baccetti, Department of Paediatrics, Obstetrics and Reproductive Medicine, Section of Biology, University of Siena, Via T. Pendola 62, 53100 Siena, Italy.
Tel: + 39-577-284173, Fax: + 39-577-233509
Email: baccetti@unisi.it
Received 2003-06-12 Accepted 2004-02-23